The biotin-(strept)avidin system is a widely used detection system in a variety of different applications (Diamandis and Christopoulos (1991) Clin. Chem. 37: 625-36). Anti-immunoglobulins, protein A, protein G, lectins, enzymes, nucleotides, nucleic acids, hormones or cells have been avidin conjugated or biotinylated and used in various applications such as immunoassays, flow cytometry, immunohistochemistry, western blots, localization of receptors, microscopy, nucleic acid hybridization, affinity chromatography, receptor-ligand interactions and hybridoma production (Diamandis Tables 1 and 2, Supra).
The avidin-biotin interaction has been used for detecting a diverse number of targets with three basic configurations. In its simplest form, avidin or streptavidin, (referred herein sometimes as (strept)avidin)) is labeled with a detectable molecule, e.g., an enzyme, fluorescent, chemiluminescent, or radioactive probe; a metal; or some other moiety. Biotin present on another reactant (antibody, nucleotide, Protein A, lectin, etc.) links the target molecule with the labeling system. This detection format is used widely for immunoassays, DNA hybridization assays, immunohistochemistry, and flow-cytometry. Avidin and streptavidin conjugates carrying a variety of detectable molecules are commercially available.
In an indirect assay, (strept)avidin is used unlabeled and serves to link the biotinylated binding agent with the biotinylated detection molecule. This variation of the system takes advantage of the multiple biotin-binding sites in each avidin or streptavidin molecule. It is also used widely in immunoassay and DNA hybridization techniques, especially with probes that can be easily biotinylated (e.g., enzymes and proteinaceous fluorescent molecules).
The third method combines the principles of the above assays to yield a more sensitive system (Hsu et al. (1981) J. Histochem. Cytochem. 29, 577-80). The concept is to mix, under controlled conditions, unlabeled (strept)avidin and a biotinylated detection reagent, e.g., an enzyme. Given the multiple biotin binding sites on (strept)avidin, one can generate a polymer having some free biotin-binding sites. This reagent is then used as in the above assays but affords superior sensitivity. Kits offering the streptavidin or avidin and biotinylated enzymes at optimized concentrations, so that one can form the complex by simply mixing the reagents, are commercially available and are known as ABC (avidin-biotin complex) kits. In U.S. Pat. No. 4,684,609, Hsu et al disclose and claim that the weight ratio of avidin to biotinylated macromolecule is from 1:1 to 16:1; 1:6 to 16:1; and 2:1 to 4:1. Table 2 of the patent discloses data obtained by using avidin to biotinylated macromolecule with ratios 32:1, 16:1, 8:1, 4:1, 2:1, 1:1, 0.5:1, and 0.25:1. The data in Table 2 of the patent further indicate that avidin to biotinylated macromolecules at ratios of 32:1, 0.5:1 and 0.25:1 did not produce a detectable signal. Also of note is that ratios of 16:1, 8:1 and 4:1 at high avidin concentrations (40 mg/ml) resulted in higher background staining. The patent mentions that a preparation of the complex formed by mixing avidin and biotinylated macromolecule will remain active for a period of several days, at least (U.S. Pat. No. 6,684,609 at co.3, lines 43-44). Thus, several ratios need to be tried, every time, to determine optimum concentrations needed for a maximum signal generation. Moreover, the successful ratios described in the patent are generally useful wherein small complexes are needed, as in immunohistochemistry. A higher amplification signal using avidin-biotin system may allow increased detection range as well as use of less primary antibodies and antigen standards, thereby reducing the costs of the assay. Thus, there is a need in the art to develop novel assays providing these and other advantages.